This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This is a subcontract with Pacific Northwest National Laboratories to characterize the comprehensive proteome of defined orthopoxvirus particles of known pathogenicity. These will include wild-type nonpathogenic vaccinia virus (VV;Western Reserve strain) and pathogenic human monkeypox virus (MPV;Zaire strain). In this objective, the protein components of the extracellular enveloped virus (EEV) and intracellular mature virus (IMV) will be determined for pathogenic MPV, and compared to that of VV. The rationale for performing these studies is that EEV particles are structurally, antigenically and biologically distinct from IMV particles. IMV particles are non-enveloped, fully infectious, extremely stable, and are the most abundant form of the virus, which makes them ideal for weaponizing, as they are well-suited to transmit infection. EEV particles are enveloped, infectious, and less stable, and are thought to mediate the long-range dissemination of virus in vivo. Elucidation of the total protein component of the two infectious particles, IMV and EEV from pathogenic and nonpathogenic isolates, will lead to new insights in orthopoxvirus morphogenesis, and will serve as a database for the identification of novel antigens for vaccine development or viral targets for the creation of small molecule inhibitors.